Flow-injection analysis immunoassays with

The use of capillaries to run immunoassay reactions was presented in the 1980s and 1990s by Halsall group [8] for the detection of atrazine. The system was based on a flow injection analysis protocol where different solutions (sample, conjugate) were injected and reacted in the capillary itself the affinity-captured enzyme conjugate then reacted with a substrate solution injected in the capillary prior to detection. The reacted substrate solution was then pushed towards an electrode for amperometric detection. The authors showed that the incubation time reached equilibrium after about 20 min, but they worked with a capillary of 360 pm diameter. In order to really reduce the incubation time below lmin, we developed disposable polymer chips with an internal diameter of 40 pm (almost one order of magnitude smaller than... 

C.S. Lim, J.N. Miller, J.W. Bridges, Automation of an energy-transfer immunoassay by using stopped-flow injection analysis with merging zones, Anal. Chim. Acta 114 (1980) 183. 

ABEI produces ECL when oxidized at 1.0 V versus Ag/AgCl in alkaline aqueous solution. In contrast to luminol, ABEI labels do not markedly lose their CL efficiency when conjugated with proteins. ECL immunoassays with a flow injection analysis (FIA) system using ABEl-isothiocyanate as a label were proposed, which have a better performance than either single-radial immunodiffusion or nephelometric immunoassays. ABEI can also be used as an oligonucleotide marker to label a DNA probe. The intensity of the ABEI ECL was linearly related to the concentration of the complementary sequence in the range 96-96 nM, and the detection limit was down to 30 pM. 

In this study two different flow-injection immunoassays are presented as well as the flexible automation system CAFCA (Computer Assisted Flow Control Analysis), which has been used for their control, uptake measurement, evaluation and visualization. Both immunoassays (a heterogeneous and a homogeneous assay) are based on the principles of flow-injection analysis and were developed for reliable, fast monitoring of relevant proteins in animal cell cultivation processes. Off-line applications of measurements of medium samples as well as online application during a mammalian cell cultivation are presented. All results are compared to results obtained with ELISA (Enzyme Linked Immunosorbend Assay). The requirements of the automation of flow-injection immunoassays with respect to their flexible control are discussed. 

A variation of flow-injection analysis, sequential-injection analysis (SIA), has recently been introduced. Contrary to flow-injection analysis, where the components are injected into a continuous flowing stream, in SIA injections are done sequentially directly into the reactor/detector system. The instrumental setup usually consists of a syringe pump in combination with an electronically controlled multi-port valve. SIA systems have been shown to be more flexible regarding injection volumes and injection times. This technology has been demonstrated to be very efficient for carrying out immunoassays. Both fluorescence and chemiluminescence immunoassays have already been carried out with this technique. 

The borderline between immunoassays performed in the flow injection analysis format and those achieved in the immunochromatographic format is sometimes difficult to establish. Flow injection renewable surface immunoassay (FIRSl) avoids the desorption step of immunochromatographic assays by using a layer of antibody-coated beads held in a jet ring cell instead of the chromatographic column. The coated beads are discarded after each analysis. To ensure assay reproducibility, antibody-coated beads with constant antigen capacity should be obtained [129,130]. 

The ICMs used for pesticide analysis include immimoassays (lAs) and the use of antibodies for sample preparation (e.g., for SPE and the cleanup of samples) [153], detection in flow-injection analysis, and biosensors. The earliest ICMs to be developed for pesticides analysis were lAs. There are various t) es of lAs, but the most frequently used in this context is the enzyme-linked immunosorbent assay (ELISA) [185]. ELISA is a heterogeneous assay because the antibodies or antigens are immobilized on a solid phase. Table 18.3 lists selected ELISA methods for the determination of pesticides in water samples [186-190]. Bjamason et al. have proposed an enzyme flow immunoassay (EFIA) using a protein G column for the determination of triazine herbicides in surface and wastewaters with a linear range between 0.1 and 10 pg/L [191]. 

Five types of amperometric detection have been applied for enzyme-based ECIA. They are flow-injection analysis with electrochemical detection (FIAEC), liquid chromatography with electrochemical detection (LCEC), amperometric detection with interdigitated array electrodes (IDA), rotating disk electrode (RDE) amper-ometry, and scanning electrochemical microscopy (SECM). Of these, the two conventional types, FIAEC and LCEC, shall be discussed in this section, leaving the discussion of the other types to Section V on miniaturized immunoassays. 

FIGURE 1.5 Schematic diagram of flow injection analysis (FIA) system with immunoassay detection RIANA (abbreviation from River ANAlyser ) based on total internal reflection fluorescence with immobilized fluorescently labeled antibodies. The source of the excitation light is a He-Ne laser, and the collected fluorescent light is filtered and detected by photodiodes (PD). (Adapted from Rodriguez-Mozaz, S. et al. 2004. Biosens. Bioelectron. 19 633-640.)... 

The combination of biosensors with flow injection analysis (FIA) techniques offers the possibility to control the whole procedure, simplifying the sequence of steps and allowing an easier optimization of the reaction conditions with reducing the measurement time by a three- to fourfold. The use in such systems of enzymatic biosensors for the determination of pesticides may provide a device competitive to immunoassay kits, or single-use disposable biosensors, due to mechanization leading to more objective measurements and efunination of operations of transfer of solutions and their volumetric measurements. 

Immunoassay is a very sensitive technique that can be applied in environmental chemistry and food chemistry, and is very often used in clinical chemistry. By inclusion of immunoassay in the flow injection system, precise control of reaction times can be achieved compatibility with any heterogeneous or homogeneous format, and detector, good accuracy, and reproducibility are assured the analysis time decreases and the analytical process becomes more flexible.243... 

Figure 3.29.A shows a flow-cell of 20 iL inner volume used to hold immobilized anti-mouse IgG bound to a rigid beaded support (activated Pierce trisacryl GF-2000). The cell was used to develop a two-site immunoassay for mouse IgG by consecutive injection of the sample, acridinium ester-labelled antibody and alkaline hydrogen peroxide to initiate the chemiluminescence, which started the reaction sequence shown in Fig. 3.29.B. Regenerating the sensor entailed subsequent injection of an acid solution, which resulted in a determination time of ca. 12 min (this varied as a fimction of the flow-rate used, which also determined the detection limit achieved, viz. 50 amol for an overall analysis time of 18 min) [218]. The sensor was used for at least one week with an inter-assay RSD of 5.9%. Attempts at automating the hydrodynamic system for use in routine analyses are currently under way.

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